Identification of new 4-(6-oxopyridazin-1-yl)benzenesulfonamides as multi-target anti-inflammatory agents targeting carbonic anhydrase, COX-2 and 5-LOX enzymes: synthesis, biological evaluations and modelling insights

Abstract Multiple inhibitions of CA, COX-2 and 5-LOX enzymes has been recognised as a useful strategy for the development of anti-inflammatory drugs that can avoid the disadvantages of using NSAIDs alone. Here, we report new pyridazine-based sulphonamides (5a-c and 7a-f) as potential multi-target anti-inflammatory candidates. First, the furanone heterocycle in the dual CA/COX-2 inhibitor Polmacoxib was replaced with the pyridazinone one. Then, a hydrophobic tail was appended through benzylation of the 3-hydroxyl group of the pyridazinone scaffold to afford benzyloxy pyridazines 5a-c. Furthermore, the structures were adorned with the polar sulphonate functionality, in pyridazine sulphonates 7a-f, that are expected to be engaged in interactions with the hydrophilic half of the CA binding sites. All of the disclosed pyridazinones were tested for inhibitory activities against 4 hCA isoforms (I, II, IX, and XII), as well as against COX-1/2, and 5-LOX. Furthermore, in vivo anti-inflammatory and analgesic effects of pyridazinones 7a and 7b were examined.


Introduction
Carbonic anhydrases (EC 4.2.1.1) are metalloenzymes that have a metal ion in their active site. 1 CAs were thoroughly studied for decades and were classified as a superfamily of enzymes with eight gene families or classes to date. 2 There are basically several cytosolic forms (CA I-III, CA VII), four membrane-bound isozymes (CA IV, CA IX, CA XII, and CA XIV), a mitochondrial form (CA V), and a secreted CA isozyme, CA VI. 3 Carbonic anhydrase is involved in diverse physiological and pathological processes including lipogenesis, gluconeogenesis, and tumorigenicity. 4 One of the major biochemical reactions involved in proton generation in resting tissues is mediated by carbonic anhydrase (CA) enzymes. 5 They are known for their ability to catalyse the reversible hydration of carbon dioxide (CO 2 þ H 2 O () H þ þ HCO 3 À ) and have also been extensively demonstrated in a plethora of physiological events at the cellular and tissue levels. 6 Numerous CA isoforms have evolved because uncatalyzed transformation is unable to meet the physiological cells' needs. 7,8 At the cellular level, overexpression of CA increases the concentration of ions (H þ and HCO 3 ), and since bicarbonate ions are necessary cellular components that are immediately recovered, local extracellular acidosis is established straight away. A decrease in tissue pH is associated with inflammatory pain, and carbonic anhydrases (CAs) are primarily responsible for proton production in tissues. 9,10 It has been well-established that the intensity of inflammation and pain-associated symptoms in diverse inflammatory disorders, such as rheumatoid arthritis, is inversely correlated with tissue pH values. 9,10 Due to their role in the regulation of pH by reversibly catalysing the conversion of CO 2 to bicarbonate and protons, CAs have been recently reported to be implicated in diverse inflammation reactions. [11][12][13] In particular, Cimaz et al. 14 reported that hCA IX and XII isoforms, which are widely expressed in hypoxic malignancies, are also over-expressed in the inflamed synovium of individuals with juvenile arthritis.
Lipid mediators, which include the classic eicosanoids, prostaglandins (PGs), and leukotrienes (LTs), are potent inflammatory mediators produced by local cell-type specific arachidonic acid metabolism. However, the arachidonic acid metabolism is carried out by the cyclooxygenase (COX) and lipoxygenase (LOX) families of enzymes, leading to the synthesis of prostaglandins and leukotrienes. Pain, inflammation, asthma, and allergies are all treated with drugs that target these enzymes. 15 Dual COX-2 and 5-LOX enzyme inhibition has recently been recognised as a useful strategy for the development of anti-inflammatory drugs that can get around the disadvantage of using NSAIDs alone. [16][17][18][19] Over the last few decades, sulphonamide-based small molecules were highlighted as an important class that is utilised for the management of many illnesses. Currently, there are more than 20 drugs in clinical usage, such as Acetazolamide (AAZ), Methazolamide, Diclofenamide, Furosemide, Bumetanide, Sulpiride, and Zonisamide ( Figure 1). It has been well-established that the sulphonamide functionality and its isosteric groups (sulfamate and sulfamide) is a crucial element for the CA and selective COX-2 inhibitory actions. 20 Polmacoxib (Acelex, Figure 2) is a sulphonamide-tethered firstin-class dual CA/COX-2 inhibitor that was approved in 2015 by the Korean Ministry of Food and Drug Safety (MFDS) for the management of osteoarthritis. [21][22][23][24] Interestingly, the CA inhibitory activity has been reported for several sulphonamide-tethered COX-2 inhibitors such as Celecoxib ( Figure 2). 23,25,26 Besides, a proof-of-concept report has revealed that the combination of the fragments of nonsteroidal anti-inflammatory drugs (NSAIDs) and CA inhibitors could improve the effectiveness of drugs used to treat ache symptoms linked to inflammatory disorders including rheumatoid arthritis (RA). 27 As a privileged scaffold, the pyridazinone motif has attracted a lot of interest in the realm of drug discovery. Due to its diverse range of biological actions and potential therapeutic uses, the pyridazinone moiety has been the focus of extensive investigations. 28 Of particular interest, diverse pyridazinone-based molecules have been reported for their anti-inflammatory [29][30][31][32][33][34][35] and carbonic anhydrase inhibitory 36,37 activities.
In this report, we developed new pyridazinone-based sulphonamide derivatives (5a-c and 7a-f, Figure 2) as potential multi-target anti-inflammatory candidates, using the multi-target strategy. [38][39][40] The hCA, COX-2, and 5-LOX enzymes, which have been implicated in the inflammatory process, are anticipated to be efficiently inhibited by the designed pyridazinone-based sulphonamides (5a-c and 7a-f). Furthermore, the in vivo antiinflammatory effects for the target pyridazinones were examined.
The design of the target pyridazinone sulphonamides is based on the bioisosteric replacement of the furanone heterocycle in the  dual CA/COX-2 inhibitor Polmacoxib with the pyridazinone one, while keeping the benzenesulfonamide motif directly attached to the heterocycle. The incorporated sulfamoyl functionality is anticipated to anchor with the catalytic Zn 2þ in the CA binding site, and also promotes COX-2 selectivity via hydrogen bonding within the hydrophilic sub-pocket in the COX-2 binding site.
In addition to the bioisosteric approach followed in the design of the new ligands, a hydrophobic tail was appended to the structure in an attempt to boost the inhibitory activity and selectivity towards hCA isoforms IX and XII. This was achieved through benzylation of the 3-hydroxyl group of the pyridazinone scaffold to afford the first set of the target benzyloxy pyridazine compounds 5a-c ( Figure 2). In addition, the structures were adorned with the polar sulphonate functionality that is expected to be engaged in interactions within the hydrophilic half of the CAs binding site. This was achieved through sulfonation of the 3-hydroxyl group with either alkyl or aromatic sulphonyl chlorides to produce the corresponding alkyl and aromatic pyridazine sulphonates 7a-b and 7c-f, respectively.

Chemistry
The synthesis of the target pyridazine derivatives is depicted in Schemes 1 and 2. A total of 10 compounds (3, 5a-c, and 7a-e) were synthesised, all of them novel molecules, not previously described in the literature. The syntheses followed known procedures previously described somewhere else. [41][42][43] The target compounds were obtained in an excellent isolated yield.
For the second set of the target pyridazine derivatives (7a-f), the synthesis was performed by reacting intermediate 3 with different aliphatic (6a-b) and aromatic (6c-f) sulphonyl chlorides in pyridine as base and solvent. The corresponding sulphonate derivatives (7a-b and 7c-f) were formed in excellent yields after stirring at 5 C for after 1h (Scheme 2).

Biological evaluation
Carbonic anhydrase inhibition Using the stopped-flow carbon dioxide hydration assay 44 all the newly synthesised pyridazines (3, 5a-c and 7a-f) reported here and the standard CA inhibitor acetazolamide (AAZ) were evaluated for their inhibitory effects against hCA isoforms (I, II, IX, and XII). The tested CA isoforms were suppressed to varying degrees by the pyridazine-based benzenesulfonamides (PBS) described here, and the inhibition data are presented in Table 1. The structureactivity relationship (SAR) was obtained based on the inhibition results as K I values for the synthesised analogues.
The cytosolic hCA I isoform was suppressed by all pyridazinetethered sulphonamide compounds (3, 5a-c and 7a-f) herein reported with inhibition constants (K I s) ranging from low to moderate nanomolar concentrations (i.e. between 23.5 nM and 362.8 nM). The in vitro kinetic data disclosed that the unsubstituted pyridazine derivative 3 is the most potent inhibitor within the tested compounds against the hCA I isoform (K I ¼ 23.5 nM), followed by pyridazines 7a and 7b (K I s ¼ 48.3 and 52.6 nM, respectively). Moderate inhibition potencies, with K I values spanning between 98.3-185.9 nM were obtained for pyridazines 5a, 5b, 7d, and 7c. Compound 7e was the least potent inhibitor (K I ¼ 362.8 nM) compared to the standard CAI AAZ (K I ¼ 250 nM). In general, both benzylation (series 5; K I : 98.3 À 221.5 nM) and sulfonation (series 7; K I : 48.3 À 362.8 nM) of the key intermediate 3hydroxy-6-oxopyridazin 3 (K I ¼ 23.5 nM) did not improve the inhibitory activities against hCA I. For the benzyloxy pyridazine series 5a-c, the substitution of the pendant phenyl motif led to a decrease in activity towards CA I (5b and 5c: K I ¼ 116.3 and 221.5 nM vs. 5a: K I ¼ 89.3 nM). For the sulphonate series 7, the alkyl sulphonates 7a and 7b (K I ¼ 48.3 and 52.6 nM, respectively) possessed better inhibitory activity than the aryl sulphonates 7c-f (K I ¼ 185.9, 123.5, 362.8 and 165.8 nM, respectively).
The synthesised pyridazine-based sulphonamide derivatives (3, 5a-c and 7a-f) all successfully suppressed the cancer-related hCA IX isoform (K I : 4.9 À 58.1 nM) as indicated in Table 1. The benzyloxy derivative 5c and the sulphonate derivative 7f were superior to the other derivatives and showed single-digit inhibitory activities (K I ¼ 4.9 and 6.4 nM, respectively) against hCA IX isoform. In addition, pyridazine derivatives 5a, 5b, 7c and 7d showed potent activity with K I values equal to 14.8, 12.3, 19.4 and 22.8 nM, respectively. It is important to emphasise that the benzyloxy-tethered derivatives 5a-c (K I : 4.9 À 14.8 nM) and the aryl sulphonate derivatives 7c-f (K I : 6.4 À 58.1 nM) displayed much enhanced inhibitory activity than the intermediate 3-hydroxy-6oxopyridazin 3 (K I ¼ 45.1 nM) against hCA IX, whereas the alkyl sulphonate derivatives 7a-b (K I ¼ 52.3 and 58.1 nM, respectively) showed less activity than compound 3.

In vitro COX-1/COX-2 and LOX inhibitory activities
In this study, further evaluation of the anti-COX-2 effect of the pyridazine-based sulphonamide derivatives (3, 5a-c and 7a-f) was carried out, in addition to evaluation of the anti-COX-1 activity in order to assess the COX-2 selectivity of the synthesised pyridazines. Celecoxib (selective COX-2 inhibitor) and Indomethacin (non-selective inhibitor) were utilised as reference COX inhibitors. IC 50 values for the inhibition of COX-1 and COX-2, as well as the selectivity index (IC 50 of COX-1/IC 50 of COX-2) are listed in Table 2. The COX-2 enzyme was effectively inhibited by the newly reported pyridazine-based sulphonamide derivatives (3, 5a-c, and 7a-f), with IC 50 values in the sub-micromolar range ranging from 0.05 to 0.14 mM, whereas COX-1 was weakly inhibited with IC 50 values spanning between 5 and 12.6 mM.
Concerning the selectivity of the target pyridazines towards COX-2 enzyme over COX-1, the findings highlighted that the pyridazine sulphonates (7a and 7b) demonstrated the highest COX-2 selectivity index of 208 and 210, respectively ( Table 2). In addition, the remaining pyridazine derivatives showed considerable selectivity towards COX-2 enzyme.
Additionally, in this study, the in vitro inhibitory activity of the pyridazine-based sulphonamide derivatives 3, 5a-c, and 7a-f against the LOX enzyme was evaluated ( Table 3).
The tested pyridazines displayed potent to moderate activity against LOX enzyme with IC 50 range of 2 -7 lM. The most potent LOX inhibitors in this work were found to be 3-hydroxy-6-oxopyridazine 3 (IC 50 ¼ 2 lM) as well as methanesulfonate 7a (IC 50 ¼ 3 lM) and ethanesulfonate pyridazine derivatives 7b (IC 50 ¼ 2.5 lM) that exerted better activity than the reference LOX inhibitor Zileuton (IC 50 ¼ 3.5 mM). It is worth noting that the best COX-2 inhibitors in this study were pyridazine sulphonates (7a and 7b) ( Table 2). As a result, both compounds 7a and 7b appear to be promising dual COX-2 and 5-LOX inhibitors.
In vivo biological evaluations Analgesic activities. The acetic acid writhing test in mice has been used in this study to examine the analgesic properties of the most potent and selective COX-2 inhibitors (pyridazine sulphonates 7a and 7b). 45 As shown in Table 4, the frequency of writhing was counted as a pain marker.
A significant reduction in the total number of writhing was observed as a direct result of treatment with both pyridazine sulphonates 7a and 7b (Number of writhing ¼ 13 and 14, respectively) in comparison to the control group (Number of writhing ¼ 40), which highlights the analgesic effect of 7a and 7b. It's interesting to note that pyridazines 7a and 7b had stronger analgesic effects than celecoxib (Table 4).
Anti-inflammatory activity. Carrageen-induced rat paw edoema protocol was exploited to evaluate the anti-inflammatory effects of the pyridazine sulphonates 7a and 7b as reported by Winter et al. 46 using celecoxib and diclofenac as reference anti-inflammatory drugs. Table 5 displays the measured percentage change of paw height.  50 values are expressed as the mean of three replicates with a standard deviation less than 10% of the mean. b Selectivity index (SI)¼ IC 50 (COX-1)/IC 50 (COX-2). Table 3. Inhibition data of LOX enzyme with pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f.
The tested molecules 7a and 7b exerted significant (p < 0.05) anti-inflammatory activities via reducing the paw height and thus reduction of the paw edoema at 3 h in comparison to the control group ( Table 5). The % inhibition of edoema of 7a and 7b were 56, and 38.5, respectively. Both compounds were superior to celecoxib with rapid onset of action after 1 h, and sustained duration until the third hour after the administration of the compound. Based on these findings, pyridazine 7a could be a good antiinflammatory candidate.
Inflammatory mediator's measurements. Based on the encouraging anti-inflammatory and analgesic properties of pyridazine sulphonates 7a and 7b stated above, an ELISA assay was used to further assess the serum level of two inflammatory mediators namely, tumour necrosis factor-alpha (TNF-a) and interleukin one beta (IL-1b).
As can be seen in Figure 3, the levels of the inflammatory mediators in the positive control group were significantly higher at the end of the paw edoema test as compared to the levels in the normal control group. On the other hand, the studied sulphonates 7a and 7b significantly lowered TNF-a and IL-1b levels when compared to the positive control group.
Ulcerogenic effects. The degree of inflammation or ulceration of the examined pyridazine sulphonates (7a and 7b) in the gastric tissues was evaluated and confirmed by histopathological examinations, following the previously described protocol. 47 As shown in Figure 4, the examination of the fasted rat stomach disclosed normal texture for pyridazine 7a as well as the reference drug celecoxib. On the other hand, pyridazine 7b displayed a mild inflammation as presented in Figure 4 (cf. red arrows). Diclofenac, also, showed inflammation and damage to the layers of the stomach (red arrows), which indicated some ulcerogenic side effects. These data suggested the safety profile of pyridazine 7a on the stomach tissues.

Molecular modelling
The most selective pyridazines against the tumour-associated isoforms CA IX and XII versus the ubiquitous cytosolic enzymes CA I and II, 5c and 7f, were investigated in silico to predict their binding mode within the active site of these four isozymes.
As expected, all docking poses showed that the benzenesulfonamide moiety bonded to the zinc ion with the deprotonated nitrogen atom of the sulphonamide group (SO 2 NH À ) in a tetracoordinated geometry. Moreover, the sulphonamide-metal coordination is stabilised by two H-bonds with the side chain OH and The value is expressed as mean ± SD (n ¼ 5). Ã Significantly different from the control group. The value is expressed as mean ± SD (n ¼ 5). Ã Significantly different from the control group. the backbone NH of T199, and by hydrophobic interactions that occur between the benzene ring and hydrophobic residues such as V143, L198 and W205 ( Figure 5). In all four enzyme isoforms (CA I, II, IX, XII), the 4-nitrophenyl moiety was oriented towards a lipophilic cleft where the mutation of some amino acid residues occurs among the different CA isoforms, is responsible for the different sizes of the pocket. Consequently, the positioning and stabilisation of the ligands are different. In the CA I, the lipophilic area is lined by Y20, L131, A132, A135, P201, P202 and Y204. Here the 4-nitrophenyl moiety of 5c engages in vdW contacts with A132, P202 and Y204, and a p-p stacking interaction with the Y204 side chain ( Figure 5(A)). In CA II the Y204L, L131F and S136Q mutations allow the NO 2 group to orient towards the Q136 and to establish an H-bond with the side chain NH 2 ( Figure 5(B)). The ligand is further stabilised by the hydrophobic contacts with F131, G132, L135 and L204 and by p-p stacking contact involving the aromatic pyridazin-3(2H)-one linker and the F131 side chain. As a result of the CA I (or II)/IX mutations at Y20V (CA I/IX), Y204A (CA II/IX) and F131V in CA IX, the aryl pendant extends towards V20 making vdW interactions with this residue and W5, P201 and P202 ( Figure 5(C)). In CA XII, the nitro group faces the same area as in CA II. However, due to the mutation Q136N (CA II/XII) and the shorter length of the amino acidic side chain, the group forms an H-bond with the side chain OH of S132 ( Figure  5(D)). The larger lipophilic pocket of the tumour-associated isoforms (CA IX and XII) compared to the off-target (CA I and II) ones, allows 5c a good fit and complementarity to the binding site, which is probably the reason for its better inhibitory profile observed experimentally.
In contrast to 5c, the tail of 7f is oriented towards the hydrophilic half of all investigated active sites. In particular, the sulphonic linker engages in an H-bond with the side chain NH 2 of the conserved residues Q92 in CA I, II, IX and XII ( Figure 5). The steric hindrance of F91 in CA I and, to less extent, F131 in CA II, led the 4-fluorophenyl group to move much closer to the hydrophilic residues N69 and E69 of the enzyme isoforms ( Figures 5(A  and B)). Moreover, the aromatic ring form p-p stacking interactions with F91 (CA I), while the pyridazin-3(2H)-one linker of 7f forms p-p stacking interaction with the F131 side chain in CA II. Similarly, the larger lipophilic area of the tumour-associated isoforms allows the aryl moiety to engage many contact points with the targets CA IX/CA XII (L91/T91, V121, V131/A131 and Q92), thus binding a larger surface as compared to the other enzyme isoforms that might account for the activity profile (i.e. CA IX ¼ CA XII > CA II ) CA I).

Conclusion
We present the design and synthesis of a novel set of pyridazinone-based sulphonamide derivatives (5a-c and 7a-f) as multi-carbonic anhydrase, COX-2, and 5-LOX inhibitors with the primary objective of developing new effective multitarget anti-inflammatory candidates. The furanone heterocycle in the dual CA/COX-2 inhibitor Polmacoxib was replaced with the pyridazinone one while keeping the benzenesulfonamide motif directly attached to the heterocycle. Then, a hydrophobic tail was appended through benzylation of the 3-hydroxyl group of the pyridazinone scaffold to afford benzyloxy pyridazine compounds 5a-c. Also, the structures were adorned with the polar sulphonate functionality, in pyridazine sulphonates 7a-f, that expected to be engaged in interactions with the hydrophilic half of all the CA active site. Most the synthesised pyridazine-based sulphonamides successfully suppressed the examined carbonic anhydrase isoforms in a variable degree; K I ranges: 23.5 À 362.8 nM, 5.3 À 106.4 nM, 4.9 À 58.1 nM and 5.3 À 49.7 nM for CA I, II, IX and XII respectively, also, they effectively inhibited the COX-2 enzyme with IC 50 values in the sub-micromolar range ranging from 0.05 to 0.14 mM, whereas COX-1 was weakly inhibited with IC 50 values spanning between 5 and 12.6 mM. In particular, the methanesulfonate and ethanesulfonate pyridazine derivatives (7a and 7b) exerted the most potent (IC 50 ¼ 0.05 and 0.06 mM) and selective (SI ¼ 208 and 210) COX-2 inhibitory activity. Moreover, the tested pyridazines displayed potent to moderate activity against LOX enzyme with IC 50 range of 2-7 lM. Further in vivo investigations for pyridazine sulphonates 7a and 7b revealed their ability to reduce the total number of writhing in mice, the rat paw edoema, and the serum levels of the inflammatory mediators (TNF-a and IL-1b), which highlights their analgesic and anti-inflammatory activities.   (20 ml). The reaction mixture was refluxed overnight, and then cooled to room temperature. The produced solid was collected by filtration, washed with hot water and dried to yield the desired compound 3 as a pale-yellow powder (92%); 1   126.05, 125.54. HRMS m/z for C 10 H 9 N 3 O 4 S [Mþ Na] þ . Calcd 290.020598, found 290.020620.
General procedure for the synthesis of benzyloxy pyridazine compounds (5a-c) The appropriate benzyl bromide derivative 4a-c (0.432 mmol) was added drop-wise to a solution of 4-(3-hydroxy-6-oxopyridazin-1(6H)-yl)benzenesulfonamide 3 (0.393 mmol) and potassium carbonate (0.432 mmol) in DMF (1 ml) at 5 C, the reaction mixture was allowed to stir for 3 h and monitored with TLC. The reaction mixture was poured into cold water and extracted with ethyl acetate (3 Â 15 ml). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure to yield pyridazines 3a-c.  General procedure for the synthesis of sulphonate pyridazine compounds (7a-f) The appropriate sulphonyl chloride derivative 6a-f (0.374 mmol) was added to a solution of 4-(3-hydroxy-6-oxopyridazin-1(6H)yl)benzenesulfonamide 3 (0.374 mmol) in pyridine (1 ml) at 5 C and stirred for 1 h. The mixture was poured into 2 N HCl solution and then extracted with ethyl acetate (3 Â 15 ml). The combined organic layers were washed with water and brine. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and evaporated under reduced pressure to yield compounds 7a-f.

COX-1, COX-2 and LOX inhibition assay
The ability of the examined pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f to inhibit both COX-1/COX-2 has been in vitro investigated by the use of COX Inhibitor screening ELISA assay kit (cat. no. 560131, Cayman, USA) according to the supplier's recommendations and as described previously. 35 Besides, LOX inhibitory screening assay kit (Cat. No. 760700, Cayman, USA) was exploited to evaluate the 5-LOX inhibitory activity of the examined pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f according to the manufacturer's instructions and as previously reported. 48 Determination of the CA inhibitory activities The experimental methodology utilised to evaluate the CA inhibitory action of the pyridazine-based sulphonamide derivatives 3, 5a-c and 7a-f disclosed here was previously described. [49][50][51][52][53] In vivo assays The experimental procedures adopted to perform the acetic acidinduced writhing assay for the analgesic activities, 45 Paw edoema assay for the anti-inflammatory activities, 46 as well as the acute ulcerogenic test 47 have been carried out as described previously.
Biochemical determination for certain inflammatory mediators ELISA assays were used to assess the serum level for the inflammatory mediators TNF-a (Kit Cat. No MBS355371, MyBioSource, USA), and IL-1b (Kit Cat. No MBS8825017, MyBioSource, USA) based on the manufacture instructions. 54

Molecular Modelling
For the computational docking study, crystal structures of CA I, II, IX and XII (PDBs: 6Y00, 3K34, 5FL4 and 5LL5, respectively [55][56][57][58] have been downloaded from Protein Data Bank 59 and prepared using the Protein Preparation Wizard tool implemented in the Schr€ odinger suite. The detailed procedures exploited in this analysis are previously reported. 54 Disclosure statement CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.